ERES function is required for assembly of the autophagy machinery immediately downstream of the Atg1 kinase complex and is associated with formation of autophagosomes at every stage of the process. ERES are core components of the autophagy machinery for the biogenesis of autophagosomes.

Large-scale proteomic analyses in Escherichia coli have documented the composition and physical relationships of multiprotein complexes, but not their functional organization into biological pathways and processes. Conversely, genetic interaction (GI) screens can provide insights into the biological role(s) of individual gene and higher order associations. Combining the information from both approaches should elucidate how complexes and pathways intersect functionally at a systems level. However, such integrative analysis has been hindered due to the lack of relevant GI data. Here we present a systematic, unbiased, and quantitative synthetic genetic array screen in E. coli describing the genetic dependencies and functional cross-talk among over 600,000 digenic mutant combinations. Combining this epistasis information with putative functional modules derived from previous proteomic data and genomic context-based methods revealed unexpected associations, including new components required for the biogenesis of iron-sulphur and ribosome integrity, and the interplay between molecular chaperones and proteases. We find that functionally-linked genes co-conserved among γ-proteobacteria are far more likely to have correlated GI profiles than genes with divergent patterns of evolution. Overall, examining bacterial GIs in the context of protein complexes provides avenues for a deeper mechanistic understanding of core microbial systems.

Comprehensive approaches to detect protein-protein interactions (PPIs) have been most successful in the yeast model system. Here we present "Cross-and-Capture," a novel assay for rapid, sensitive assessment of PPIs via pulldown of differently tagged yeast strain arrays. About 500 yeast genes that function in DNA replication, repair, and recombination and nuclear proteins of unknown function were chromosomally tagged with six histidine residues or triple VSV epitopes. We demonstrate that the assay can interrogate a wide range of previously known protein complexes with increased resolution and sensitivity. Furthermore, we use "Cross-and-Capture" to identify two novel protein complexes: Rtt101p-Mms1p and Sae2p-Mre11p. The Rtt101p-Mms1p interaction was subsequently characterized by genetic and functional analyses. Our studies establish the "Cross-and-Capture" assay as a novel, versatile tool that provides a valuable complement for the next generation of yeast proteomic studies.

Acinetobacter baumannii, a Gram-negative coccobacillus, is notorious for its involvement in opportunistic infections around the world. Its resistance to antibiotics makes treatment of infections challenging. In this study, we describe a novel response regulator protein, AvnR (A1S_2006) that regulates virulence-related traits in A. baumannii ATCC17978. Sequence analysis suggests that AvnR is a CheY-like response regulator and contains the RNA-binding ANTAR (AmiR and NasR transcription anti-termination regulators) domain. We show that AvnR plays a role in regulating biofilm formation (on glass and plastic surfaces), surface motility, adhesion to A549 cells as well as in nitrogen metabolism in A. baumannii . RNA-Seq analysis revealed that avnR deletion results in altered expression of more than 150 genes (116 upregulated and 42 downregulated). RNA-Seq data suggest that altered biofilm formation and surface motility observed in the avnR deletion mutant is likely mediated by previously unknown pathways. Of note, was the altered expression of genes predicted to be involved in amino acid transport and metabolism in avnR deletion mutant. Biolog phenotypic array showed that deletion of avnR hampered A. baumannii ATCC17978’s ability to metabolize various nitrogen sources, particularly that of glutamic acid, serine, histidine, aspartic acid, isoleucine and arginine. Taken together our data show that AvnR, the first ANTAR protein described in A. baumannii, affects virulence phenotypes as well as its ability to metabolize nitrogen sources.

Legionella pneumophila is a facultative intracellular parasite of freshwater protozoa that undergoes a complex multiphasic lifecycle (Mondino et al., 2020;Newton et al., 2010). Two major morphological forms feature predominantly: a replicative rod-shaped form and a motile coccoid-shaped mature intracellular form (MIF) developed upon egress from the spent protozoan host (Garduño et al., 2002;Greub & Raoult, 2003;Robertson et al., 2014). This MIF (henceforth referred to as cyst form) features distinct ultrastructural characteristics (thickened cell wall, multilayer membrane laminations, poly-3-hydroxybutyrate [PHB] inclusions) ideal for long-term survival between protozoan hosts (Faulkner & Garduño, 2002). It is currently viewed that inhalation of aerosolized cyst-laden water droplets by a susceptible individual leads to infection of alveolar macrophages and manifestation of Legionnaires' disease.Successful infection of protozoa and macrophages by L. pneumophila is enabled by the Dot/Icm Type IV Secretion System (T4SS) which translocates >300 effector proteins into the host cell to manipulate diverse cellular processes (Burstein et al., 2016;Finsel & Hilbi, 2015;Gomez-Valero et al., 2019). In macrophages, the main targets include abrogation of the phagosome-lysosome fusion pathway and